Compositions and methods for the manufacture of lipid nanoparticles

ABSTRACT

The invention relates to methods, processes and apparatuses for the manufacture of lipid nanoparticles having a therapeutic payload.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/023,919, filed on Mar. 22, 2016, which is a is a35 U.S.C. § 371 national stage filing of International Application No.PCT/US2014/056984, filed on Sep. 23, 2014, which in turn, claims thebenefit of priority to U.S. Provisional Patent Application No.61/881,630, filed on Sep. 24, 2013. The entire contents of each of theforegoing applications are incorporated herein by reference.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled121301_08203_SL.txt created on Dec. 10, 2018 which is 789 bytes in size.The information in electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to systems and processes for the manufacture oflipid nanoparticles effective to deliver a nucleic acid payload,specifically RNAi agents.

BACKGROUND OF THE INVENTION

Double-stranded RNA molecules (dsRNA) have been shown to modulate geneexpression in a highly conserved mechanism known as RNA interference(RNAi). This mechanism has now become the focus for the development of anew class of pharmaceutical agents for treating disorders that arecaused by the aberrant or unwanted regulation of a gene.

Given the focus in the art surrounding delivery of RNAi therapeutics,effective delivery of therapeutic compounds to a target organ or systemis often the largest hurdle facing a potentially lifesaving treatment.And while certain methods of formulating therapeutics in lipid particlesand liposomes are known in the art, for example those described in U.S.Pat. Nos. 7,901,708; 7,811,603; 7,030,097; 6,858,224; 6,106,858;5,478,860 and 5,908,777, the contents of which are each incorporatedherein by reference, there remains a need for improved processes andapparatuses for the manufacture of lipid nanoparticles capable ofcarrying a therapeutic payload. The present invention provides suchmethods, processes and systems for the manufacture of lipidnanoparticles which sufficiently encapsulate a nucleic acid payload,specifically RNAi agents, for delivery to mammalian cells.

SUMMARY OF THE INVENTION

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

In one embodiment is provided a method of preparing a formulationcomprising lipid nanoparticles comprising an RNAi agent payload.According to this method a first solution is mixed with a secondsolution in a mixing connector. The first solution comprises anethanolic solution comprising one or more lipids and having a totallipid concentration of approximately 30 mg/mL and the second solutioncomprises citrate buffered aqueous solution comprising one or more RNAiagents and having an RNAi agent concentration of approximately 1 mg/mLand a pH of between 3 and 6. The mixture is then diluted in a vesselcontaining a buffer solution thereby producing a formulation comprisinglipid nanoparticles comprising an RNAi agent payload. The buffer may beany suitable buffer and is preferably citrate buffer or PBS.

Mixing in the connector may occur at a linear flow rate of between about300,000 cm/hr to about 2,500,000 cm/hr for each solution, independently.The volume ratio of the first solution to the second solution may bebetween 1:2 and 1:5, preferably 1:3.

Also contemplated as within the invention is a system for themanufacture of a formulation comprising lipid nanoparticles comprisingan RNAi agent payload. This system comprises a first reservoir providinga first solution, a second reservoir providing a second solution, afirst pump, operably connected to said first reservoir and configured toregulate the flow of said first solution at a linear flow rate and asecond pump, operably connected to said second reservoir and configuredto regulate the flow of said second solution at a linear flow rate. Thesystem also contains a mixing connector comprising at least a firstinlet, a second inlet and an outlet, wherein said first inlet receivesflow from said first pump and said second inlet receives flow from saidsecond pump, at least one heat exchanger operably connecting each ofsaid first and said second pumps to said inlets of the mixing connector,respectively, and a vessel for receiving effluent from the outlet ofsaid mixing connector.

The methods and systems of the present invention are useful in themanufacture of lipid nanoparticles for formulating an RNAi agentpayload, wherein the RNAi agent is selected from the group consisting ofsiRNA, dsRNA, miRNA, and nucleotide sequences encoding the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the configuration of the manufacturing andultrafiltration system of one embodiment of the present invention.

DETAILED DESCRIPTION

The present invention describes a process for the manufacture andpreparation of formulations of RNAi agents, particularly smallinterfering ribonucleic acids (siRNAs) in lipid nanoparticles (LNPs).The process involves mixing of ethanolic solution of lipids with abuffered aqueous solution of siRNA and the downstream processing of thatmixture.

Two major driving forces lead to the formation of LNPs and theencapsulation of the the nucleic acid payload (e.g., siRNA) in thisprocess; first, a sharp decrease of the solubility of the lipids as aresult of mixing with the aqueous solution (the lipids are soluble inethanol and have very low solubility in water) and second, the chargeinteraction between the positively charged ionizable lipid and thenegatively charged sugar-phosphate backbone of the siRNA.

There are four general steps in the process: (1) solution preparation,(2) mixing, thereby resulting in creation of the formulations, (3)ultrafiltration, and (4) final concentration adjustment. Theultrafiltration step includes an initial concentration, a diafiltrationto remove the ethanol and exchange the buffer, and final concentration.Generally, the lipids are dissolved in ethanol (200 proof) to reach apredetermined ratio and a total lipid concentration of approximately 30mg/mL and the RNAi agent, e.g., siRNA is dissolved in an aqueous buffer(e.g., citrate buffer, 10 mmol, pH 4) to a concentration ofapproximately 1 mg/mL. Pumping the two solutions with controlled linearflow rates and a volume ratio Lipid/RNAi agent of approximately 1:3 intoa mixing connector and diluting the mixture approximately 5-fold bycollecting it into a vessel containing predetermined amount of PBSallows for the formation of the lipid nanoparticle formulations withconcurrent encapsulation of the nucleic acid payload, e.g., siRNA.Additional removal of the ethanol and exchange of the citrate bufferwith PBS using an ultrafiltration (UF) step leads to the final drugproduct with the desired lipid and drug concentration.

Preparation and/or Manufacture of Lipid Nanoparticle Formulations

The process flow diagram for the preparation of lipid nanoparticleshaving a nucleic acid payload is presented in FIG. 1.

In one aspect, the invention relates to a system for the manufacture oflipid nanoparticles 100. The manufacturing system may be coupled,directly or indirectly to an ultrafiltration and concentrationadjustment system 200.

In some embodiments, pre-reservoirs are provided for each solution. Inone embodiment, a pre-reservoir 10 feeds into a reservoir for theethanolic lipid solution 20. The ethanolic lipid solution is preparedusing ethanol (200 proof) as a solvent to approximately 30 mg/mL totallipid concentration.

Likewise a pre-reservoir 11 feeds into a reservoir for buffered aqueoussolution 21. Purified house water may be used as a solvent for theaqeous buffered solution preparation to approximately 1 mg/mL siRNA incitrate buffer at pH 4. The exact concentrations of the two solutionsmay be determined by any means known in the art. For example, this maybe done using analytical HPLC methods prior to the mixing step.

Optionally, provided prior to each of the reservoirs 20 and 21 is one ormore filters 60. The filters may be of any type but preferably are0.45/0.2 μm filters.

Operably connected by tubing 70 to each of said first and secondreservoirs is a pump 30. Pumps may include peristaltic or positivedisplacement. Any of several pumps may be used in the present invention.In one embodiment the pumps for each reservoir are the same. In oneembodiment the pumps used are PrepStar SD-1 Titanium pumps with eitheran 800 mL/min or 3200 mL/min pump head (Agilent/Varian Part NoR007105050).

In the present invention, the pumps may be operated at different flowrates of between 100 mL/min to 3200 mL/min using the systems describedherein. It is to be understood that depending on the tubing chosen, theflow rate in mL/min may vary. However, the flow rates contemplated bythe invention independent of choice of tubing include linear flow ratesfor the ethanolic solution of about 300,000 cm/hr to about 900,000cm/hr. Linear flow rates for the buffered solution may be from about1,500,000 to about 2,120,000 cm/hr.

In one embodiment, the linear flow rate of the ethanolic lipid solutionis between 100-300 mL/min (between 303,133 cm/hr-909,400 cm/hr),preferably 200 mL/min (606,267 cm/hr).

In one embodiment, the linear flow rate of the buffered aqueous solutionof RNAi agent is between 500-700 mL/min (1,515,665 cm/hr-2,121,931cm/hr), preferably 600 mL/min (1,818,801 cm/hr).

The tubings (flow lines) and fittings of the system of the invention maybe of any suitable material. PEEK tubing with various internal diameters(ID) and outer diameters (OD) are provided herein.

Mixing of the two solutions occurs when each is connected to a pump 30and pumped through a heat exchanger 40 to the mixing connector 50. Inone embodiment the ethanolic lipid solution is pumped in tubing (flowline) 80 at a linear flow rate of approximately 200 mL/min and theaqueous buffered solution is pumped in tubing (flow line) 80 at a linearflow rate of approximately 600 mL/min. The exact flow rates of the twopumps are calculated based on the exact concentration of the twosolutions and the target lipid/RNA w/w ratio (10:1).

According to the present invention, the linear flow rates for theethanolic lipid solution may range from 100-300 mL/min and the linearflow rates for the aqueous buffered solution may range from 500-700mL/min. The optimal linear flow rates are achieved by the combination ofthe pumps volume flow rates (mL/min) and the ID of “Tubing 2” 80. At theconcentrations described the approximate Pump 1(lipid)/Pump 2 (RNA)ratio is 1:3.

Several pumps were evaluated for suitability in this process. HPLC typepumps were chosen for the accuracy of the volume delivered as well asfor their capability to withstand high back pressures. As describedabove, the lipid/RNA ratio is determined by the total lipidconcentration in the ethanolic solution, the RNA concentration in theaqeous buffered solution as well as by the ratio of the flow rates ofthe two pumps. Because of the precision of the HPLC pumps, theflow-rate-ratio can be controlled very tightly. As such, targetedlipid/RNA ratios of 10:1 w/w and 14:1 w/w were achieved with highaccuracy using the same solutions just by adjusting the pump flow rates.

According to the present invention, heat exchangers 40 are positionedbetween each of the pumps and at least one inlet of the mixing connector50 via tubing 80. The mixing connector may be of any suitable polymer orstainless steel. It may be of the T-shape or Y-shape form. The mixingconnector may have 2 or more inlets and the inlets may be configuredregularly or irregulary and be connected to a single outlet.

The effluent from the outlet of the mixing connector 50 then flows viatubing 90 into a vessel 95 where a dilution of the formulation isachieved with PBS. According to the present invention, the ethanolconcentration in reservoir 95 or 96 may be from 1 to 5%, 1%, 2%, 3%, 4%or 5%, or any value within the range of 1-5%. In one embodiment, theethanol concentration is ≤5% in reservoir 95 or 96. The dilution may be1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9× or 10× or more. In one embodiment,the dilution is 5×.

The process is evaluated by the lipid and RNA concentration in the finalbulk, the lipid to RNA ratio, the degree of RNA encapsulation and theparticle size, dispersity and distribution. According to the presentinvention, particle size may range from 50 to 100 nm with a PDI ofbetween 0.02 to 0.10. Favorable particle sizes are those of between 60nm and 80 nm with a PDI of less than 0.10.

It is understood that temperature may affect the lipid mixture physicalstate and as such affect the outcome of the process.

Turning back to FIG. 1, the present invention also includes anultrafiltration system 220 either directly or indirectly connected toreservoir 95 or 96 containing the formulations. The goal for theultrafiltration step is to remove the ethanol and exchange the citratebuffer with PBS 210. The ultrafiltration process consists of threesteps: concentration, diafiltration and concentration adjustment. Thefirst step is one of concentration during which the solution isconcentrated about 5-fold. During this concentration step, the ethanoland buffer concentrations do not change but the particle concentrationis increased. Second is a diafiltration step, during which the ethanoland the citrate buffer salts are removed and exchanged with PBS. In thisstep, 10 volume exchanges are used with the permeate (waste) beingremoved to vessel 230. Third is a final concentration step during whichthe concentration of the particles is brought up to the equivalent of2.5-3 mg/mL RNA.

Critical for the ultrafiltration step 220 are the choice of the pump,material of the cassettes, retentate flow rate, membrane area, andtransmembrane pressure. A rotary lobe pump (Sartorius) or a diaphragmpump may be used for the step, along with polyethersulfone (PES)cassettes from Sartorius (Part 305 14668 01E SW). Diaphragm pumps mayalso be used in the ultrafiltration step.

The final concentration during the UF step results in a bulk productwith approximately 2.5-3.0 mg/mL RNA concentration in vessel 240. Theexact concentration is established using an HPLC analytical method andthe concentration is adjusted to 2 mg/mL by diluting the bulk with PBS210 to vessel 250. The bulk product may be filtered 260 and stored in avessel 280 at 2-8° C.

Lipid Nanoparticle Payload

According to the present invention, the process and apparatus disclosedare useful in the preparation and manufacture of lipid nanoparticlescarrying a therapeutic payload, specifically a nucleic acid payload.Therapeutic payloads include proteins, peptides, nucleic acids, smallmolecules, antibodies and the like.

The nucleic acid payload may include RNAi agents (e.g. siRNA, dsRNA,miRNA) as well as antisense molecules, ribozymes, and plasmid-basedconstructs or any nucleic acid based molecules. As used herein a“therapeutic payload” is any compound, substance or molecule which has atherapeutic benefit and which can be incorporated into or encapsulatedwithin a lipid nanoparticle made by the methods described herein.

As used herein, the term “RNAi agent” refers to an agent that containsRNA as that term is defined herein, and which mediates the targetedcleavage of an RNA transcript or target sequence via an RNA-inducedsilencing complex (RISC) pathway.

As used herein, the term “RNAi agent mix” or “RNAi agent cocktail”refers to a composition that comprises more than one RNAi agent.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature, but also analogs and derivatives of RNAcomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to anRNAi agent that includes an RNA molecule or complex of molecules havinga hybridized duplex region that comprises two anti-parallel andsubstantially complementary nucleic acid strands, which will be referredto as having “sense” and “antisense” orientations with respect to atarget RNA.

The term “antisense strand” or “guide strand” refers to the strand of anRNAi agent, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches may be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an RNAi agent that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

The duplex region can be of any length that permits specific degradationof a desired target RNA through a RISC pathway, but will typically rangefrom 9 to 36 base pairs in length, e.g., 15-30 base pairs in length.Considering a duplex between 9 and 36 base pairs, the duplex can be anylength in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,or 36 and any sub-range therein between, including, but not limited to15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs,15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs,15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs,18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs,19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs,19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs,20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs,21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs,21-23 base pairs, or 21-22 base pairs.

The two strands forming the duplex structure can be from a single RNAmolecule having at least one self-complementary region, or can be formedfrom two or more separate RNA molecules. Where the duplex region isformed from two strands of a single molecule, the molecule can have aduplex region separated by a single stranded chain of nucleotides(herein referred to as a “hairpin loop”) between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure. The hairpin loop can comprise at least one unpairednucleotide; in some embodiments the hairpin loop can comprise at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 20, at least 23 or more unpaired nucleotides.Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than a hairpin loop, the connecting structure is referredto as a “linker.” The term “siRNA” is also used herein to refer to adsRNA as described above.

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA.

In yet another embodiment, the RNA of an RNAi agent, e.g., a dsRNA orsiRNA, is chemically modified to enhance stability or other beneficialcharacteristics. The nucleic acids featured in the invention may besynthesized and/or modified by methods well established in the art, suchas those described in “Current protocols in nucleic acid chemistry,”Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y.,USA, which is hereby incorporated herein by reference. Modificationsinclude, for example, (a) end modifications, e.g., 5′ end modifications(phosphorylation, conjugation, inverted linkages, etc.) 3′ endmodifications (conjugation, DNA nucleotides, inverted linkages, etc.),(b) base modifications, e.g., replacement with stabilizing bases,destabilizing bases, or bases that base pair with an expanded repertoireof partners, removal of bases (abasic nucleotides), or conjugated bases,(c) sugar modifications (e.g., at the 2′ position or 4′ position) orreplacement of the sugar, as well as (d) backbone modifications,including modification or replacement of the phosphodiester linkages.

Another modification of the RNA of an RNAi agent featured in theinvention involves chemically linking to the RNA one or more ligands,moieties or conjugates that enhance the activity, cellular distributionor cellular uptake of the RNAi agent. Such moieties include but are notlimited to lipid moieties such as a cholesterol moiety, peptides,peptidomimetics, vitamins and the like.

In some embodiments, the RNAi agents formulated in the lipidnanoparticles comprise pharmaceutical compositions. As used herein, a“pharmaceutical composition” comprises a pharmacologically effectiveamount of an RNAi agent formulated in a lipid nanoparticle. As usedherein, “pharmacologically effective amount,” “therapeutically effectiveamount” or simply “effective amount” refers to that amount of an RNAiagent effective to produce the intended pharmacological, therapeutic orpreventive result. For example, if a given clinical treatment isconsidered effective when there is at least a 10% reduction in ameasurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 10%reduction in that parameter. For example, a therapeutically effectiveamount of an RNAi agent can reduce gene protein levels by at least 10%or more.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of genes. In general, asuitable dose of RNAi agent will be in the range of 0.01 to 200.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 to 50 mg per kilogram body weight per day. Forexample, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition maybe administered once daily, or the RNAi agent may be administered astwo, three, or more sub-doses at appropriate intervals throughout theday or even using continuous infusion or delivery through a controlledrelease formulation.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual RNAi agents encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more RNAi agent compounds and (b) one ormore biologic agents which function by a non-RNAi mechanism. The RNAiagent may be formulated in the lipid nanoparticles of the presentinvention while the non-RNAi agent may be separately formulated. In oneembodiment, the two are formulated together in a lipid nanoparticle.

Lipid Nanoparticles

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an RNAi agent or aplasmid from which an RNAi agent is transcribed. SNALPs are described,e.g., in U.S. Patent Application Publication Nos. 20060240093,20070135372, and in International Application No. WO 2009082817. Theseapplications are incorporated herein by reference in their entirety.

As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683.

The lipid nanoparticles of the present invention typically have a meandiameter of about 50 nm to about 150 nm, more typically about 60 nm toabout 130 nm, more typically about 70 nm to about 110 nm, most typicallyabout 60 nm to about 80 nm, and are substantially nontoxic. In addition,the nucleic acids when present in the nucleic acid-lipid particles ofthe present invention are resistant in aqueous solution to degradationwith a nuclease.

In one embodiment, the lipid to drug ratio (mass/mass ratio; w/w ratio)(e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 toabout 50:1, from about 1:1 to about 25:1, from about 10:1 to about 14:1,from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid may comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

Lipid-dsRNA nanoparticles typically form spontaneously upon mixing.Depending on the desired particle size distribution, the resultantnanoparticle mixture can be extruded through a polycarbonate membrane(e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, suchas Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusionstep can be omitted. Ethanol removal and simultaneous buffer exchangecan be accomplished by, for example, dialysis or tangential flowfiltration. Buffer can be exchanged with, for example, phosphatebuffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0,about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which is hereinincorporated by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). LNP01 formulationsare described, e.g., in International Application Publication No. WO2008/042973, which is hereby incorporated by reference.

Other formulations may incorporate XTC, MC3, ALNY-100 or C12-200.

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10,2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S.Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and InternationalApplication No. PCT/US2010/022614, filed Jan. 29, 2010, which are herebyincorporated by reference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, and International Application No.PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated byreference.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal.

The total RNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated RNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total RNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” RNA content (as measured by thesignal in the absence of surfactant) from the total RNA content. Percententrapped RNA is typically >85%. For SNALP formulation, the particlesize is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm,at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, atleast 110 nm, and at least 120 nm. The suitable range is typically aboutat least 50 nm to about at least 110 nm, about at least 60 nm to aboutat least 100 nm, or about at least 50 nm to about at least 80 nm.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the RNAi agents and methods featured in theinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

EXAMPLES Example 1. RNAi Agent Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Oligonucleotide Synthesis

All oligonucleotides are synthesized on an AKTAoligopilot or OligoPilot400 synthesizers. Commercially available controlled pore glass solidsupport (dT-CPG, 500 {acute over (Å)}, Prime Synthesis) and RNAphosphoramidites with standard protecting groups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O-N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeare purchased from (Promega). All phosphoramidites are used at aconcentration of 0.15M in acetonitrile (CH₃CN) except for2′-O-methyluridine, which is used at 0.15M concentration in 10% THF/ANC(v/v). Coupling/recycling time of 16 to 23 minutes is used. Theactivator is 5-ethyl thiotetrazole (0.6M, American InternationalChemicals); for the PO-oxidation iodine/water/pyridine is used and forthe PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence is performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled RNAi agents are synthesized from the corresponding Quasar-570(Cy-3) phosphoramidite are purchased from Biosearch Technologies.Conjugation of ligands to 5′-end and or internal position is achieved byusing appropriately protected ligand-phosphoramidite building block. Anextended 15 min coupling of 0.1 M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid-support-bound oligonucleotide. Oxidation of theinternucleotide phosphite to the phosphate is carried out using standardiodine-water as reported (1) or by treatment with tent-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate is introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent. The cholesterol phosphoramidite is synthesized in house andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mLglass bottle (VWR). The oligonucleotide is cleaved from the support withsimultaneous deprotection of base and phosphate groups with 80 mL of amixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C.The bottle is cooled briefly on ice and then the ethanolic ammoniamixture is filtered into a new 250-mL bottle. The CPG is washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixtureis then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dryice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionis then quenched with 50 mL of 20 mM sodium acetate and the pH isadjusted to 6.5. Oligonucleotide is stored in a freezer untilpurification.

Analysis

The oligonucleotides are analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phasepreparative HPLC. The unconjugated oligonucleotides are purified byanion-exchange HPLC on a TSK gel column packed in house. The buffers are20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodiumphosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides are pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotidess arediluted in water to 150 μL and then pipetted into special vials for CGEand LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

RNAi Agent Preparation

For the general preparation of RNAi agents, equimolar amounts of senseand antisense strand are heated in 1× PBS at 95° C. for 5 min and slowlycooled to room temperature. Integrity of the duplex is confirmed by HPLCanalysis.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1. It will beunderstood that these monomers, when present in an oligonucleotide, aremutually linked by 5′-3′-phosphodiester bonds.

TABLE 1 Abbreviations of nucleotide monomers Abbreviation Nucleotide(s)A Adenosine C Cytidine G Guanosine T Thymidine U Uridine N anynucleotide (G, A, C, T or U) a 2′-O-methyladenosine c2′-O-methylcytidine g 2′-O-methylguanosine U 2′-O-methyluridine dT2′-deoxythymidine S phosphorothioate linkage

Example 2. Preparation of Solutions

Ethanolic Lipid Solution and Buffered Aqueous RNAi Agent Solution

The ethanolic solution in this example contains ionizable lipid,PEG-conjugated lipid, DSPC, and cholesterol and the buffered aqueoussolution contains the siRNA in pH 4 citrate buffer. The following lipids(Table 2) were used to make the AF-011 premix. The structures of theseare shown in Table 3 along with their average molecular weights.

TABLE 2 Components of Ethanolic Lipid Solution (AF-011) ComponentGrams/L Mole % MC3 17.122 50 DSPC 4.214 10 Cholesterol 7.939 38.5PEG-DMG 2.044 1.5 Total 31.320 100

TABLE 3 Lipids Molecular Weight Lipid (g/mole) Chemical Name andStructure DLin-MC3- DMA  642.09

PEG₂₀₀₀-C- DMG 2555*

DSPC  790.16

Cholesterol  386.65

To make one liter of AF-011, the following procedure was followed.

In a clean and sterile 500 mL glass bottle, add 7.939 g cholesterol, add400 mL Absolute Ethanol (Pharmco-AAPER, 200 proof, anhydrous, ACS/USPGrade, Catalog #111000200), seal bottle with Teflon coated cap and heatwith shaking at 50° C. until dissolved. In a clean and sterile 250 mLglass bottle, add 4.214 g DSPC, add 200 mL Ethanol, seal bottle withTeflon coated cap and heat with shaking at 40° C. until dissolved. In aclean and sterile 100 mL glass bottle, add 2.044 g PEG-DMG, add 100 mLEthanol, seal bottle with Teflon coated cap and heat with shaking at 40°C. until dissolved. In a clean and sterile 100 mL glass bottle, add17.122 g MC3, add 100 mL Ethanol, seal bottle with Teflon coated cap andheat with shaking at 40° C. until dissolved. Once all lipid componentsare dissolved, transfer each to a clean 1 L graduated cylinder rinsingwith ethanol. Adjust the volume to 1 L with ethanol. Filter solutionthrough a 0.2 μm Nylon bottle-top filter.

The components and concentrations for the preparation of the ethanoliclipid solution and the buffered aqueous solution of RNAi agent aresummarized in Error! Reference source not found. and Error! Referencesource not found. below. Ethanol (200 proof) was used as a solvent forthe lipid solution and purified house water was used as a solvent forthe RNAi agent preparation. Both solutions were filtered through0.45/0.2 μm filters prior to use. The lipid solution was prepared toapproximately 30 mg/mL total lipid concentration and the RNAi agentsolution contained approximately 1 mg/mL siRNA (RNAi agent) in citratebuffer at pH 4. The exact concentrations were determined using HPLCanalytical HPLC methods prior to the mixing step.

TABLE 4 AF-011 Components and Concentrations Component ManufacturerProduct Number Grams/L Mole % MC3 Genzyme LP-04-203 17.122 50 DSPCLipoid 18:0/18:0 4.214 10 Cholesterol Sigma SyntheCholC1231 7.939 38.5PEG-DMG Sunbright 161G981V700 2.044 1.5 Ethanol (200 PHARMCO- 111000200solvent solvent Proof) AAPER Total 31.320 100

The control duplex, AD-1955, which targets the luciferase gene has thesense sequence cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 1) and the antisensesequence UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 2), where lower casenucleotides are modified by 2′Omethyl and dT stands for deoxyThymidineand “s” represents a phosphorothioate linkage.

TABLE 5 RNAi agent Solution Components and Concentration Product TotalComponent Manufacturer Number Grams/L # of L Grams Sodium SAFC W3026001.105 3 3.315 Citrate Citric Acid Sigma-Aldrich 251275 1.310 3 3.93 RNAiagent, Alnylam AD-1955 1.044 3 3.132 AD-1955

A 4 L graduated cylinder was charged with 2.5 L water and a stir baradded for mixing on a stir plate. 3.315 g sodium m citrate, 3.93 gcitric acid and 3.132 g AD-1955 were added to the stirring water. Thecomponents were stirred until completely dissolved and the pH checked.The volume was adjusted to 3 L with water and stirring continued for 10minutes. The solution was filtered through the bottle-top filter andcollected in a 5 L glass media bottle. The solution was stored at 4° C.until ready for use.

The formulation with the AF-011 Pre-mix (31.32 mg/mL) and 1 mg/mLsiRNA/10 mM Sodium citrate pH4 solution were mixed at a volume ratio of3:1 (RNAi agent:lipid) siRNA to AF-011 to give a desired Lipid/siRNA w/wratio of 10 to 14.

Additional formulations which may be prepared according to the presentinvention include those listed in Table 5B.

TABLE 5B Formulations cationic lipid/non-cationic lipid/cholesterol/Cationic PEG-lipid conjugate Name Lipid Lipid:siRNA ratio SNALP DLinDMADLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1S-XTC XTC XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 AF-05 XTC XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 AF-06 XTC XTC/DSPC/Cholesterol/PEG-DMG57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 AF-07 XTCXTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~6:1 AF-08 XTCXTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA ~11:1 AF-09 XTCXTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1 AF-10ALN100 ALN100/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1AF-011 MC3 MC-3/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1AF-012 C12-200 C12-200/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA 10:1 AF-013 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5Lipid:siRNA: 33:1 AF-014 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5Lipid:siRNA: 11:1 AF-015 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG50/10/35/4.5/0.5 Lipid:siRNA: 11:1 AF-016 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA: 7:1 AF-017 MC3 MC3/DSPC/Chol/PEG-DSG50/10/38.5/1.5 Lipid:siRNA: 10:1 AF-018 MC3 MC3/DSPC/Chol/PEG-DMG50/10/38.5/1.5 Lipid:siRNA: 12:1 AF-019 MC3 MC3/DSPC/Chol/PEG-DMG50/10/35/5 Lipid:siRNA: 8:1 AF-020 MC3 MC3/DSPC/Chol/PEG-DPG50/10/38.5/1.5 Lipid:siRNA: 10:1 AF-021 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 AF-022 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

-   DLinDMA: 1,2-Dilinolenyloxy-N,N-dimethylaminopropane-   XTC: 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane-   ALN100:    (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine-   C12-200: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl    4-(dimethylamino)butanoate (MC3)-   1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol-   DSPC: distearoylphosphatidylcholine-   DPPC: dipalmitoylphosphatidylcholine-   PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with    avg mol wt of 2000)-   PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg    mol wt of 2000)-   PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg    mol wt of 2000)

Example 3. Instrumentation

In one embodiment, the process of preparing lipid nanoparticles havingan RNAi agent payload includes a mixing system operating in tandem withan ultrafiltration system. One configuration of each system is outlinedin Tables 6 and 7. These two systems are also shown in FIG. 1.

TABLE 6 Mixing Equipment Manufacturer Product number Note Pump 1Agilent/Varian PrepStar SD-1 Titanium 200 mL/min* Lipid Solution 800mL/min - R007105050 3200 mL/min - R007105050 Pump 2 Agilent/VarianPrepStar SD-1 Titanium 600 mL/min* RNAi agent 800 mL/min - R007105050solution 3200 mL/min - R007105050 Heat Exergy 00540-02 Shell & Tube 23Exchanger 1 series and 2 Mixing Swagelok ⅛″-SS-200-3 Stainless Steel T-Connector connector Option 1 Mixing GE 18-1170-59 Plastic Y-connectorConnector Option 2 Tubing 1 Teflon; OD 5/16″; ID ¼″ Tubing 2IDEX/Upchurch Peek Natural; #1534 ID 0.062″ Solution A 200 mL/min -616,085 cm/h Solution B 600 mL/min - 1,848,255 cm/h Tubing 3IDEX/Upchurch Peek Natural; #1544 ID 0.08″ Filter Sartorius 5441307H5-00Sartopore 2 300, Sterile Capsule Filter, 0.45 + 0.2 μm *Both Pump 1 andPump 2 are the same type of pumps capable of pumping up to 800 mL/min

TABLE 7 Ultrafiltration Equipment Manufacturer Product Number Note SliceSystem Sartorius LabTop rotary lobe pump PES cassettes Sartorius 30514668 01 E SW 100 kDa PBS Ambion AM9625

Example 4. Tubings and Fittings

In the present invention, various types of tubings and fittings wereinvestigated for optimal performance in several systems. These arelisted in Table 8.

TABLE 8 Tubing and Fittings Description Item Part # ID″ OD″ ID (cm) Area(cm²) PEEK tubing Gray 1565 0.015 0.0625 0.0381 0.001140092 Orange 15320.02 0.0625 0.0508 0.00202683 Green 1533 0.03 0.0625 0.0762 0.004560367Natural 1538 0.04 0.0625 0.1016 0.00810732 Natural 1537 0.055 0.06250.1397 0.015327901 Natural 1534 0.062 0.125 0.15748 0.019477835 Natural1544 0.08 0.125 0.2032 0.032429279 TEEs, Crosses and Manifolds Tee P-632TEE, WITH P-245/P-200N, 1/16 IN, ¼-28, .020 IN (.5 mm) THRU, TEFZEL ®(ETFE) Tee P-712 TEE, WITH P-200/P-235, 1/16 IN, ¼-28, .020 IN (.5 mm)THRU HOLE, PEEK ™ Tee P-714 TEE, WITH P-200/P-235, 1/16 IN, ¼-28, .040IN (1.02 mm) THRU HOLE, PEEK ™ Tee P-727 TEE, WITH F-300, 1/16 IN,10-32, .020 IN (.5 mm) THRU HOLE, PEEK ™ Tee P-728 TEE, WITH F-300, 1/16IN, 10-32, .05 IN (1.25 mm) THRU HOLE, PEEK ™ Static U- MIXING TEE,STATIC WITH 3 F-300, HIGH Mixing 466 PRESSURE, PEEK ™ WITH 10 μm UHMWPEFRIT Tee Cross P-634 CROSS, WITH P-245/P-200N, 1/16 IN, ¼-28, .020 IN(.5 mm) THRU, TEFZEL ® (ETFE) Cross P-722 CROSS, WITH P-200/P-235, 1/16IN, ¼-28, .020 IN (.5 mm) THRU HOLE, PEEK ™ Cross P-723 CROSS, WITHP-300/P-335, ⅛ IN, ¼-28, .05 IN (1.25 mm) THRU HOLE, PEEK ™ ManifoldP-150 MANIFOLD, 7-PORT, ¼-28 FOR 1/16 IN OD TUBING Manifold P-151MANIFOLD, 7-PORT, ¼-28 FOR ⅛ IN OD TUBING Manifold P-170 MANIFOLD,7-PORT, 10-32 FOR 1/16 IN OD TUBING Manifold P-190 MANIFOLD, 9-PORT,¼-28 FOR ⅛ IN OD TUBING Manifold P-191 MANIFOLD, 9-PORT, ¼-28 FOR 1/16IN OD TUBING Mixing Connectors Y P-512 Y CONNECTOR, WITH P-235/P-200,1/16 IN, ¼- Connector 28, .020 IN (.5 mm) THRU HOLE, PEEK ™ Y P-513 YCONNECTOR, WITH P-335/P-300, ⅛ IN, ¼- Connector 28, .040 IN THRU HOLE,PEEK ™ Y P-514 Y CONNECTOR, WITH P-335/P-300, ⅛ IN, ¼- Connector 28,.060 IN THRU HOLE, PEEK ™ Y P-515 Y CONNECTOR, WITH P-133/P-132, 3/16IN, 5/16- Connector 24, .125 IN THRU HOLE, PEEK ™ Micro- P-470 SplitterValve

Example 5. Instrumentation for the Manufacture of Lipid Nanoparticle(LNP) Formulations with siRNA: AKTA Oligopilot 100

An AKTA system was configured to deliver buffered aqueous siRNAsolutions through the A-Pump and the Lipid pre-mix solution (ethanol)through the B-Pump. After the pumps, the PEEK tubing (Orange, PN1532,1/16″OD×0.02″ID) came to a TEE (P-728) with the outlet tubing (TFZL1/16″OD×0.04″ID) directed to a tube for collection of formulations. Fourexperiments were performed:

Experiment 1: Formulation 5-15

Flow A pump=15 mL/min. Flow B pump=5 mL/min.

Experiment 2: Formulation 10-30

Flow A pump=30 mL/min. Flow B pump=10 mL/min.

Experiment 3: Formulation 20-60

Flow A pump=60 mL/min. Flow B pump=20 mL/min.

Experiment 4: Formulation 30-90

Flow A pump=90 mL/min. Flow B pump=30 mL/min.

Particle size (Zavg; d·nm) and dispersion (PDI; particle dispersionindex) were determined using a Zetasizer from Malvern Instruments;Zetasizer Nano-ZS, Model #: ZEN3600, Serial #: MAL1028752. Particlesize, Zavg, in the Experiments ranged from 98.2-478 for Experiment 1;101-118 for Experiment 2; 104-137 for Experiment 3 and 131-166 forExperiment 4. Particle size dispersion was found to be from 0.142-0.557for Experiment 1; 0.19-0.262 for Experiment 2; 0.246-0.386 forExperiment 3 and 0.303-0.411 for Experiment 4.

Example 6. Instrumentation for the Manufacture of Lipid Nanoparticle(LNP) Formulations with siRNA: AKTA Oligopilot 100 with Small TEE

Various TEE sizes were investigated in the AKTA system. These are listedin Table 9. Again, particle size and dipersion were determined using aZetasizer from Malvern Instruments.

TABLE 9 Size and Dispersion Z-Ave Sample Name (d · nm) PdIST-ORANGE-5/15 107 0.202 ST-ORANGE-10/30 109.1 0.137 ST-ORANGE-20/6087.83 0.113 ST-ORANGE-30/90 87.75 0.151 ST-GREEN-5/15 128.3 0.217ST-GREEN-10/30 111 0.139 ST-GREEN-20/60 97.25 0.123 ST-GREEN-30/90 1070.138 ST-NAT04-5/15 114.4 0.169 ST-NAT04-10/30 91.79 0.156ST-NAT04-20/60 98.8 0.107 ST-NAT04-30/90 105.2 0.103 ST-GREY/NAT04-128.4 0.172 5/15 ST-GREY/NAT04- 116.5 0.151 10/20 ST-GREY/NAT04- 106.10.097 20/60 ST-GREY/NAT04- 93.38 0.142 30/90

Example 7. Instrumentation for the Manufacture of Lipid Nanoparticle(LNP) Formulations with siRNA: Two Waters Prep-LC Systems Side by Side

In another embodiment, the siRNA solution (diluted 10 fold with citratebuffer) was attached to the Waters Prep-LC-300 and the AF-011 lipidsolution (diluted 10 fold with ethanol) was attached to the WatersPrep-LC-150. The outlet of each system was attached to a TEE (P-728) byPEEK tubing (several sizes were investigated) with the outlet tubing(TFZL 1/16″OD×0.04″ID) directed to a 50 mL Falcon tube prepped with 15mL 1× PBS for collection of formulations. Various configurations wereinvestigated and these are described here.

Experiment 1: Here three different types of 1/16″OD PEEK tubing weretested: Orange (1532, 0.02″ID), Green (1533, 0.03″ID), and Natural(1537, 0.055″ID). For each tubing, five different flow rates (Lipid(mL/min.)/siRNA (mL/min.)) were tested. These were 5/15, 30/90, 45/135,60/180, 100/300. Fifteen (15) mL of formulation was collected in eachtube prepped with 15 mL 1× PBS. Samples were allowed to sit at 4° C.overnight then measured for particle size and dispersion with theZetasizer. The data are shown in Table 10.

TABLE 10 Size and dispersion using the Waters system Z-Ave Sample (d ·nm) PDI Orange5/15 217.2 0.346 Orange30/90 129.7 0.06 Orange45/135 90.70.053 Orange60/180 88.67 0.07 Orange100/300 90.33 0.1 Green5/15 91.790.163 Green30/90 95.53 0.056 Green45/135 87.05 0.124 Green60/180 88.340.073 Green100/300 91.2 0.115 Nat055-5/15 151.8 0.104 Nat055-30/90 87.510.052 Nat055-45/135 85.57 0.031 Nat055-60/180 95 0.003 Nat055-100/30091.65 0.131

Experiment 2: In a second experiment using the same system, threedifferent TEEs were tested. These included (1) Large Tee (LT), P-728,(2) Small Tee (ST), P-727 and (3) Mixing Tee (MT), U-466. For each TEEtested, three different PEEK Tubing sizes were also investigated. Theseincluded (1) Orange (1532), 0.02″ID, (2) Green (1533), 0.03″ID and (3)Natural (1538), 0.04″ID. Finally, for each TEE and Tubing, fivedifferent flow rates (Lipid (mL/min.)/siRNA (mL/min.)) were tested.These included (1) 5/15, (2) 30/90, (3) 45/135, (4) 60/180, and (5)100/300.

Fifteen (15) mL of the formulation was collected in each tube preppedwith 15 mL 1× PBS. Samples were allowed to sit at 4° C. overnight thenmeasured for particle size and dispersion with the Zetasizer. The dataare shown in Table 11.

TABLE 11 Size and dispersion using the Waters system Z-Ave Sample Name(d · nm) PdI LT-Orange-5/15 125.7 0.074 LT-Orange-30/90 91.43 0.02LT-Orange-45/135 95.82 0.109 LT-Orange-60/180 80.81 0.061LT-Orange-100/300 89.61 0.125 LT-Green-5/15 86.07 0.067 LT-Green-30/9084.73 0.041 LT-Green-45/135 82.2 0.043 LT-Green-60/180 88.61 0.077LT-Green 100/300 93.69 0.092 LT-Nat04-5/15 85.57 0.054 LT-Nat04-30/9089.1 0.033 LT-Nat04-45/135 88.53 0.064 LT-Nat0460/180 87.06 0.073LT-Nat04-100/300 87.63 0.016 ST-Orange-5/15 81.98 0.042 ST-Orange-30/9071.78 0.026 ST-Orange-45/135 76.39 0.064 ST-Orange-60/180 87.5 0.138ST-Orange-100/300 89.37 0.019 ST-Green-5/15 83.46 0.068 ST-Green-30/9080.5 0.057 ST-Green-45/135 79.47 0.083 ST-Green-60/180 88.91 0.066ST-Green100/300 97.96 0.152 ST-Nat04-5/15 75.31 0.025 ST-Nat04-30/9076.04 0.062 ST-Nat04-45/135 72.51 0.074 ST-Nat04-60/180 83.32 0.058ST-Nat04-100/300 91.52 0.081 MT-Orange-5/15 71.36 0.065 MT-Orange-30/9080.09 0.06 MT-Orange 45/135 85.79 0.167 MT-Orange-60/180 81.91 0.112MT-Orange-100/300 84.39 0.192 MT-Green-5/15 73.73 0.033 MT-Green-30/9075.45 0.135 MT-Green-45/135 72.94 0.131 MT-Nat040-5/15 70.57 0.118MT-Nat04-30/90 70.82 0.063 MT-Nat04 45/135 74.81 0.077 MT-Nat04-60/18072.82 0.201 MT-Nat04100/300 84.21 0.148

Particles with favorable size, between 60-80 nm and PDI of less than 0.1were observed with the Waters system.

Example 8. Instrumentation for the Manufacture of Lipid Nanoparticle(LNP) Formulations with siRNA: Bench-Top Prep LC

In one embodiment, the system was modified with the siRNA on the A-Pump(Pump 2 of Table 6) and the AF-011 on the B-Pump (Pump 1 of Table 6). Itwas reasoned that a method that delivers 25% B will give the correct 3:1liquid RNAi agent:Lipid ratio for mixing and should give a lipid/siRNAratio of 10 to 14. Flow rate, tubing size and TEE size will were variedto define the optimal process. Both AF-011 and siRNA solutions werediluted 10 fold.

In the present system, three different TEES were tested. These included(1) Large Tee (LT), P-728, (2) Small Tee (ST), P-727 and (3) StainlessSteel Tee (SST), Swagelok ⅛″. For each TEE, five different PEEK Tubingsizes were tested. These included (1) Orange (1532), 0.02″ID, (2) Green(1533), 0.03″ID, (3) Natural (1538), 0.04″ID, (4) Nat ⅛″ (1534),0.062″ID, and (5) Nat ⅛″ (1544), 0.08″ID. For each TEE and Tubing, fourdifferent flow rates (Lipid (mL/min.)/siRNA (mL/min.)) were tested.These included (1) 30/90, (2) 60/180, (3) 150/450 and (4) 200/600.

Fifteen (15) mL of the formulation was collected in each tube preppedwith 15 mL 1× PBS. Samples were allowed to sit at 4° C. overnight thenmeasured for particle size and dispersion with the Zetasizer. The dataare shown in Table 12.

TABLE 12 Size and dispersion data using the bench top system Z-AveSample Name (d · nm) PdI ST-ORANGE-30/90 71.8 0.057 ST-ORANGE-60/18078.56 0.08 ST-ORANGE-150/450 79.02 0.084 ST-GREEN-30/90 68.68 0.059ST-GREEN-60/180 78.34 0.076 ST-GREEN-150/450 81.27 0.116ST-GREEN-200/600 85.05 0.115 ST-NAT04-30/90 80.55 0.1 ST-NAT04-60/18079.91 0.091 ST-NAT04-150/450 84.97 0.198 ST-NAT04-200/600 84.15 0.193ST-NAT02-30/90 74.28 0.059 ST-NAT02-60/180 72.62 0.131 ST-NAT02-150/45087.54 0.135 ST-NAT02-200/600 89.05 0.157 ST-NAT08-30/90 87.84 0.092ST-NAT08-60/180 98.69 0.099 ST-NAT08-150/450 76.98 0.121ST-NAT08-200/600 81.06 0.174 LT-ORANGE-30/90 69.25 0.064LT-ORANGE-60/180 68.23 0.108 LT-ORANGE-150/450 74.93 0.067LT-GREEN-30/90 74.3 0.118 LT-GREEN-60/180 74.58 0.113 LT-GREEN-150/45082.07 0.192 LT-GREEN-200/600 83.18 0.129 LT-NAT04-30/90 74.58 0.076LT-NAT04-60/180 83.63 0.067 LT-NAT04-150/450 94.24 0.17 LT-NAT04-200/600103.5 0.334 LT-NAT062-30/90 74.83 0.069 LT-NAT062-60/180 73.8 0.081LT-NAT062-150/450 87.51 0.07 LT-NAT062-200/600 76.27 0.121LT-NAT08-30/90 74.15 0.089 LT-NAT08-60/180 70.97 0.125 LT-NAT08-150/45076.35 0.075 LT-NAT08-200/600 80.47 0.108 SST-NAT062-30/90 89.82 0.094SST-NAT062-60/180 75.29 0.069 SST-NAT062-150/450 73.87 0.055SST-NAT062-200/600 68.19 0.053 SST-NAT08-30/90 96.37 0.076SST-NAT08-60/180 76.37 0.029 SST-NAT08-150/450 71.71 0.038SST-NAT08-200/600 70.58 0.088

Superior performance was observed with the Natural 0.062″ID tubing andthe Stainless Steel TEE.

Example 9: Instrumentation Variations

Several experiments were repeated which gave particles <75 nm and PDI<0.01 in Example 8. In addition, these experiments were run using siRNAin Sodium Acetate buffer as opposed to the Citrate buffer used in allprevious experiments. The data are shown in Tables 13-15.

TABLE 13 buffer data CITRATE Tubing d (cm) mL/min cm/h Z PDI ST-ORANGE0.0508 30/90 888086/2664259 71.8 0.057 ST-GREEN 0.0762 30/90394705/1184115 68.68 0.059 ST-NAT062 0.15748 30/90 92412/277238 74.280.059 LT-ORANGE 0.0508 30/90 888086/2664259 69.25 0.064 LT-ORANGE 0.0508150/450 4440431/13321295 74.93 0.067 LT-NAT04 0.1016 30/90222021/666064  74.58 0.076 LT-NAT062 0.15748 30/90 92412/277238 74.830.069 LT-NAT062 0.15748  60/180 184825/554476  73.8 0.081 LT-NAT080.2032 30/90 55505/166516 74.15 0.089 SST-NAT062 0.15748 150/450462063/1386190 73.87 0.055 SST-NAT062 0.15748 200/600 616084/184825468.19 0.053 SST-NAT08 0.2032 150/450 277526/832580  71.71 0.038SST-NAT08 0.2032 200/600 370035/1110107 70.58 0.088

TABLE 14 Citrate buffer data CITRATE Tubing d (cm) mL/min cm/h Z PDIST-ORANGE 0.0508 30/90 888086/2664259 77.07 0.076 ST-GREEN 0.0762 30/90394705/1184115 69.16 0.077 ST-NAT062 0.15748 30/90 92412/277238 69.560.061 LT-ORANGE 0.0508 30/90 888086/2664259 70.77 0.062 LT-ORANGE 0.0508150/450 4440431/13321295 NA NA LT-NAT04 0.1016 30/90 222021/666064 72.74 0.062 LT-NAT062 0.15748 30/90 92412/277238 72.91 0.021 LT-NAT0620.15748  60/180 184825/554476  68.79 0.106 LT-NAT08 0.2032 30/9055505/166516 69.85 0.064 SST-NAT062 0.15748 150/450 462063/1386190 70.740.027 SST-NAT062 0.15748 200/600 616084/1848254 72.86 0.028 SST-NAT080.2032 150/450 277526/832580  68.45 0.035 SST-NAT08 0.2032 200/600370035/1110107 71.67 0.048

TABLE 15 Sodium acetate buffer data NaAc Tubing d (cm) mL/min cm/h Z PDIST-ORANGE 0.0508 30/90 888086/2664259 63.5 0.05 ST-GREEN 0.0762 30/90394705/1184115 56.07 0.141 ST-NAT062 0.15748 30/90 92412/277238 55.010.127 LT-ORANGE 0.0508 30/90 888086/2664259 60.91 0.07 LT-ORANGE 0.0508150/450 4440431/13321295 NA NA LT-NAT04 0.1016 30/90 222021/666064  60.30.248 LT-NAT062 0.15748 30/90 92412/277238 63.19 0.055 LT-NAT062 0.15748 60/180 184825/554476  59.8 0.121 LT-NAT08 0.2032 30/90 55505/16651661.45 0.083 SST-NAT062 0.15748 150/450 462063/1386190 63.59 0.201SST-NAT062 0.15748 200/600 616084/1848254 65.43 0.243 SST-NAT08 0.2032150/450 277526/832580  58.39 0.141 SST-NAT08 0.2032 200/600370035/1110107 56.09 0.192

Example 10: Modified System with Stainless Steel Tee and Sodium Acetatevs. Citrate

In another test, the Stainless Steel TEE (SST) with ⅛″OD tubing sized0.062″ID and 0.08″ID at flow rates of 150/450 and 200/600 with 25 mMNaOAc (sodium acetate), 10 nM NaOAc (sodium acetate), and 10 mM sodiumcitrate were tested. Flow rates listed are lipid solution:RNAi agentsolution as with Example 9. The data are shown in Table 16.

TABLE 16 Stainless Steel Tee data SST Tubing d (cm) mL/min cm/h Z PDINaOAc25-Nat062 0.15748 150/450 462063/1386190 70.22 0.212 NaOAc25-Nat0620.15748 200/600 616084/1848254 65.69 0.215 NaOAc25-Nat08 0.2032 150/450277526/832580  62.39 0.174 NaOAc25-Nat08 0.2032 200/600 370035/111010755.77 0.205 NaOAc10-Nat062 0.15748 150/450 462063/1386190 73.99 0.089NaOAc10-Nat062 0.15748 200/600 616084/1848254 77.92 0.039 NaOAc10-Nat080.2032 150/450 277526/832580  85.08 0.046 NaOAc10-Nat08 0.2032 200/600370035/1110107 73.46 0.138 Citrate10-Nat062 0.15748 150/450462063/1386190 80.78 0.026 Citrate10-Nat062 0.15748 200/600616084/1848254 87.19 0.046 Citrate10-Nat08 0.2032 150/450 277526/832580 85.42 0.082 Citrate10-Nat08 0.2032 200/600 370035/1110107 81.62 0.074

Example 11: Modified System with Stainless Steel Tee, Citrate, TubingConfiguration and Ratio

The modified system was further evaluated to test the Stainless SteelTEE (SST) with ⅛″OD tubing sized 0.062″ID, 0.08″ID and mixed sizes atflow rates of 150/450 and 200/600 with 10 mM sodium citrate. Further,the RNAi agent:Lipid ratio was changed to 5:1 siRNA/Lipid mixing. Theresults are shown in Table 17.

TABLE 17 Stainless Steel Tee and varied ratio data SST Tubing diameterTubing d (cm) mL/min cm/h Z PDI Citrate-62/62-1 0.15748 150/450462063/1386190 64.8 0.041 Citrate-62/62-1 0.15748 200/600 616084/184825466.31 0.079 Citrate-80/62-1 0.2032/0.15748 150/450 277526/832580  66.220.05 Citrate-80/62-1 0.2032/0.15748 200/600 370035/1110107 67.24 0.012Citrate-80/80-2 0.15748 150/450 462063/1386190 66.31 0.081Citrate-80/80-2 0.15748 200/600 616084/1848254 63.5 0.082Citrate-62/62-2 0.2032 150/450 277526/832580  63.98 0.046Citrate-62/62-2 0.2032 200/600 370035/1110107 62.12 0.058Citrate-80/62-2 0.2032/0.15748 150/450 462063/1386190 63.91 0.072Citrate-80/62-2 0.2032/0.15748 200/600 616084/1848254 65.82 0.052CitrateDilute-80/80- 0.15748 100/600 185018/1848254 62.67 0.076100/600-2 CitrateDilute-62/62- 0.2032 100/600 370035/1110107 61.72 0.047100/600-2 CitrateDilute-80/62- 0.2032/0.15748 100/600 370035/1110107.62.57 0.058 100/600-2

Example 12: Modified System Small Scale

Small scale studies were then performed on the modified system with0.075 g/L AD-1955 in 10 mM Citrate on Pump-A and AF-011 diluted 10 foldwith ethanol on Pump-B. The tubing from the pumps to SST were 0.062″IDand the mixing connector outlet tubing was 0.08″ ID. Prime lines wereset to flow 600 mL/min at 25% B. The stream was collected in a 5 Lbottle with 1.5 L 1× PBS until 5 L total volume. Ultrafiltration (UF) ontandem UF with 2 Hydrosart 100K Slice of a Slice (Sartorius biotech).The solution was then concentrated to 200 mL then diafiltered with 2 L1× PBS. The flow was reversed to get all of the formulation in chamber.The formulation was collected. Some visible particles were observed.Attempts to filter through a 0.2 μm bottle top filter failed because ofclogging. Syringe filtering also failed after a few mL's. Particle sizeand dispersion was measured by the Zetasizer. Measuring by Horiba lightscattering particle size distribution analyzer (Horiba Scientfic) showedlarge aggregates after UF that were removed by filtering through a 0.2μm syringe filter. The data are shown in table 18.

TABLE 18 Particle size and dispersion Z-Ave Sample Name (d · nm) PdIpermeate1 0 0.077 Initial Batch 55.28 0.059 Batch1-130 56.81 0.068Batch1-230 50.64 0.043 Batch1-330 50.28 0.074 Batch1-430 52.18 0.07Batch1-530 52.08 0.084 Batch1-730 51.03 0.073 Batch1-1030 56.22 0.168Batch1-1030-2 55.13 0.132 Batch1-fin-filter 54.24 0.06 Batch1-finnofilter 55.85 0.094 Batch1-fin-filter 53.6 0.054

Given the larger aggregate particles observed in the test,ultrafiltration options were further investigated using the systemdefined in Table 19. Inlet and outlet tubing refers to the inlet andoutlet of the mixing connector.

TABLE 19 Small Scale Batch 2: Ultrafiltration System Modified-Varianpump Inlet Tubing ID″ Nat 0.062 Outlet Tubing Nat 0.08 TEE StainlessSteel Total Flow 600 mL/min. Flow A 450 mL/min. Flow B 150 mL/min.AD-1955 10 fold dilution AF-011 10 fold dilution

The stream was collected in a 5 L bottle with 1 L 1× PBS until finalvolume was 3.5 L. 1 L was set aside in the coldroom. Ultrafiltration wasperformed using a Labtop with 100K Hydrosart Slice Cassette. The vesselwas filled with 1× PBS, followed by concentration of the formulationwith a pump set to 190 RPM. After concentration, the formulation wasexchanged with 1× PBS. The final 425 mL was collected and particle sizeand dispersion was measured with the Zetasizer. The data are shown intable 20.

TABLE 20 Particle size and dispersion: Hydrosart ultrafiltrationHydrosart Z-Ave Sample Name (d.nm) PdI mix-3 52.79 0.037 Conc-t1 57.230.009 Conc-t2 56.81 0.043 Conc-t3 51.88 0.071 PBS-t1 60 0.046 PBS-t258.09 0.064 PBS-t3 59.87 0.083 PBS-t4 60.84 0.104 PBS-final 61.03 0.136final2 63.7 0.143 filtered 62.16 0.12

Following from this run, 1 L of the material previously set aside in thecoldroom was subjected to ultrafiltration with 100K PES Slice Cassetteat 190 RPM. The formulation was concentrated and exchanged with 1× PBSthen 575 mL was collected. The flow of the permeate was measured and thedata are shown in Table 21. Particle size and dispersion are shown intable 22.

TABLE 21 Permeate flow rate mL/min. Measure Point Permeate Initial 166Final Conc. 76 PBS1 68 PBS2 66 PBS3 64 PBS4 64 PBS5 56 PBS Final 68

TABLE 22 Particle size and dispersion: PES ultrafiltration PES Z-AveSample Name (d · nm) PdI initial 54.46 0.063 conc-t1 53.32 0.028 t2 530.033 pbs-t0 52.96 0.05 pbs-t1 53.14 0.014 pbs-t2 50.07 0.013 pbs-t351.24 0.071 pbs-t4 50.27 0.053 conc575 50.46 0.087

Subsequently, 575 mL from the sample above was subjected toultrafiltration using a smaller bench top device but with the same samecassette used on the larger labtop and concentrated to 50 mL. Particleswere collected and measured. The data are shown in Table 23.

TABLE 23 Particle size and dispersion: PES concentrated PES-concentratedZ-Ave Sample Name (d · nm) PdI conc300 52.73 0.079 conc100 52.78 0.094Final conc 52.79 0.1 filter 51.86 0.101 reverse 52.77 0.123

Example 13. Variation of Lipid Concentration and Temperature

In an effort to define the optimal lipid concentration and temperatureconditions, the modified system with 0.075 g/L AD-1955 in 10 mM Citrateon Pump-A and AF-011 diluted 10 fold with ethanol on Pump-B was used.The tubing from the pumps to SST were 0.062″ID and the mixing connectoroutlet tubing was 0.08″ ID. Lipid concentrations were 1×, 3×, 6× and 10×the RNAi agent. Temperature was varied from 10° C. to 35° C.Formulations were created and particles measured for size anddispersity. The lipid:RNAi data are shown in Tables 24 and 25, and theTemperature data are shown in Tables 26 and 27.

TABLE 24 Particle size and dispersion Lipid to RNAi agent multiplier forlipid/RNAi Z-Ave agent flow rate of 150/450 (d · nm) PdI 1X 66.33 0.0193X 67.06 0.028 6X 69.54 0.074 10X  80.15 0.06

TABLE 25 Particle size and dispersion Lipid to RNAi agent multiplier forlipid/RNAi Z-Ave agent flow rate of 200/600 (d · nm) PdI 1X 66.82 0.0083X 70.96 0.089 6X 79.5 0.111 10X  80.69 0.143

TABLE 26 Temperature study Temperature for lipid/RNAi agent Z-Ave flowrate of 150/450 (d · nm) PdI 10° C. 77.58 0.107 15° C. 76.34 0.069 25°C. 76.43 0.056 30° C. 76.02 0.074 35° C. 77.09 0.089

TABLE 27 Temperature study Temperature for lipid/RNAi agent Z-Ave flowrate of 200/600 (d · nm) PdI 10° C. 69.49 0.048 15° C. 73.96 0.046 25°C. 70.4 0.05 30° C. 76.32 0.077

Example 14. Heat Exchanger Study

The modified system of Table 28 was fitted with Series Exergy 23 Shellin Tube Heat Exchangers on both the A and B pump lines between the pumpand the mixing connector. Heat Exchanger temperature was controlled byJulabo Circulating heating/cooling bath (Julabo Labortechnik GmbH).Temperatures of 25° C. through 45° C. were then tested.

TABLE 28 System Configuration A: Pump AD-1955, 1.5 L B: Pump AF-011lipid mixture, 0.5 L Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat0.08  TEE (mixing connector) Stainless Steel

TABLE 29 Particle size and dispersion for flow rate Lipid: RNAi agent200/600 Z-Ave 200/600 (d · nm) PdI 25° C. 67.88 0.072 30° C. 70.43 0.07435° C. 70.78 0.064 40° C. 70.16 0.071 45° C. 73.83 0.08

TABLE 30 Particle size and dispersion for flow rate Lipid: RNAi agent150/450 Z-Ave 150/450 (d · nm) PdI 25° C. 74.9 0.051 30° C. 72.7 0.07235° C. 75.03 0.124 40° C. 74.8 0.089 45° C. 76.02 0.112

Proceeding with a flow rate of 200/600 and a temperature of 25° C., thesystem outlined in Table 31 was coupled to the ultrafiltration systemoutlined below in Table 32 and the permeate flows were measured.Particle size and dispersion values for this system configuration weremeasured and are shown in Table 33.

TABLE 31 System Configuration A: Pump AD-1955, 1.5 L B: Pump AF-011lipidmixture, 0.5 L Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat 0.08 TEE (mixing connector) Stainless Steel Total Flow 800 mL/min. Flow A 600mL/min. Flow B 200 mL/min. Temperature ° C. 25

TABLE 32 Ultrafiltration System Configuration Vessel Temp 15° C. PumpSpeed 150 RPM Measure Point mL/min. Permeate Initial 234 1.5 L 165 3.5 L132 5.5 L 128 7 L 156 8 L 146 9 L 138 10 L 136 11 L 136 12 L 130 13 L125

TABLE 33 Particle size and dispersion Sample Z-Ave Name (d · nm) PdIInitial Mix 66.8 0.068 Conc. 1 L 67.53 0.048 Conc. 3.5 L 67.29 0.081Conc. 5 L 66.97 0.09 Conc. 7 L 67.18 0.057 Conc. 8.5 L 66.78 0.085 Conc.Final 66.89 0.07 PBS 1.5 L 65.31 0.081 PBS 3 L 65.51 0.058 PBS 4 L 65.570.087 PBS 5 L 65.37 0.086 PBS 6 L 65.31 0.048 Final 67.37 0.078 Final65.39 0.07 Filtered Rev Flow 67.02 0.071

Example 15. Mixing Connector Study

On the system outlined in Table 35 below, th pumps were primed to wasteand then the formulation was collected in a 10 L Bottle with 6 L 1× PBS(8 L total formulation).

In this run, the objective was to test the Y and T shaped mixingconnectors (GE, PN-18-1170-59, lot 4465564) in place of the stainlesssteel Tee (SST) at 800 mL/min. Both symmetrical and asymmetricalconfigurations were explored. Collection was in a 50 mL Falcon Tubeprepped with 15 mL 1× PBS. Permeate flow rate was measured and thesedata are shown in Table 35. Particle size was then measured. These dataare in Table 36.

Ultrafiltration and 1× PBS Exchange was performed on Labtop Systemfitted with 3×100K PES Slice cassettes. The system was cleaned in placewith ethanol wash followed by water wash then equilibrated with 1× PBS.Once equilibrated and with the vessel full with 1× PBS, the formulationwas added by vacuum and concentrated to 500 mL then diafiltered with 5 L1× PBS. The final formulation was collected and filtered through a Pall0.2 μm PES capsule filter.

TABLE 34 System configuration A: Pump AD-1955, 1.5 L B: Pump AF-011lipidmixture, 0.5 L Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat 0.08 TEE Stainless Steel, Poly Y Total Flow 800 mL/min. Flow A 600 mL/min.Flow B 200 mL/min. Temperature ° C. 25

TABLE 35 Permeate flow rate Vessel Temperature ° C. 7 Pump Speed Initial= 250 RPM, increase to 300 RPM Permeate amount Permeate flow rate(mL/min.) 3 L 400 4 L 280 7.5 L 210 8.4 L 188

TABLE 36 Particle size and dispersity Sample Name Z-Ave (d · nm) PdIInitial 68.26 0.062 Final 65.92 0.095 FinalFilter 67.81 0.023 PolyYS73.03 0.13 PolyYAS 78.18 0.063 Batch Final Filter 66.64 0.08

Example 16. Pump Speed and Permeate Flow Rate in Ultrafiltration

On the system outlined in Table 37 below, the pumps were primed to wasteand then the formulation was collected in a 10 L Bottle with with 3 L 1×PBS (5.667 L total formulation).

In this run, the objective was to make mall scale formulations at 22% B,24% B and 26% B in 50 mL Falcon Tubes.

TABLE 37 System configuration A: Pump, Varian AD-1955, 2 L B: Pump,Varian AF-011lipid mixture, 0.667 L Inlet Tubing ID″ Nat 0.062 OutletTubing ID″ Nat 0.08 TEE Stainless Steel Total Flow 800 mL/min. Flow A600 mL/min. Flow B 200 mL/min. Temperature ° C. 25

Ultrafiltration and 1× PBS exchange was performed on a On Labtop Systemthat has been fitted with 5×100K PES Slice cassettes. The system wascleaned in place with ethanol wash followed by water wash thenequilibrated with 1× PBS. Once equilibrated and with the vessel fullwith 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 300 RPM. The flow rate declined rapidly. Thepump speed was increased to 550 RPM which did not help the permeateflow. The cassettes seemed to be clogged. Concentration was stopped with1.5 L remaining that was saved at 4° C. for an UF experiment withdifferent conditions. The data are shown in Table 38.

TABLE 38 Pump Speed and Permeate flow rate Vessel Temperature ° C. 7Pump Speed Initial = 250 RPM, increase to 550 RPM Pump Speed Permeateflow rate (mL/min.) 300 RPM, initial 500 350 RPM 80 400 RPM 50 550 RPM,TMP set to 18 75 After concentrating 1.5 L 48 After concentrating 2 L 48

The remaining 1.5 L of formulation saved from the previous run wasdiluted to 3.5 L with 1× PBS. The UF system was set up with 3×100K PEScassettes, cleaned and equilibrated with 1× PBS. The formulation wasconcentrated to 500 mL and diafiltered with 5 L 1× PBS. The initial pumpspeed was 303 RPM and was increased to 400 RPM after concentratingbefore starting the PBS exchange. The data are shown in Table 39.

TABLE 39 Ultrafiltration Permeate Flow rate Vessel Temperature ° C.  7Initial Permeate flow rate 1000 (mL/min.) Conc 1 L 600 Conc 2.5 L 400Conc 4 L 330 Conc 5 L 360 Conc 6 L 330 PBS XC 1 L 290 PBS XC 2 L 280 PBSXC 4 L 270 PBS XC 7 L 240

Particle size and dispersity from this experiment are shown in Table 40and in Table 41.

TABLE 40 Particle size and dispersity Lipid/siRNA Sample Name Z-Ave (d ·nm) PdI Ratio initial-mix 75.94 0.07 8.3 UF1 80.38 0.073 6.9 UF2 80.570.068 7.8 UF3 94.48 0.205 8.6 final-UF 95.41 0.209 9.7

TABLE 41 Particle size and dispersity Sample Name Z-Ave (d · nm) PdIdilute-mix 75.72 0.041 final-UF 76.59 0.085

The particle distribution data from the Horiba analysis showed that theparticles from the initial mix and the second ultrafiltration (UF) weregood and there is a large distribution and larger particles present.

Example 17. Pump Speed and Permeate Flow Rate in Ultrafiltration:Temperature Study

On the system defined in Table 42, the pumps were primed to waste andthen the formulations were collected in 10 L Bottle with 6 L 1× PBS(7.71 L total formulation).

In this run, two formulations were made at 27% B. (1) Batch 4.1 at 25°C. and (2) Batch 4.2 at 40° C.

TABLE 42 System configuration A: Pump, Varian AD-1955 (1.25 L per batch)B: Pump, Varian AF-011 lipid mixture (0.46 L L per batch) Inlet TubingID″ Nat 0.062 Outlet Tubing ID″ Nat 0.08 TEE Stainless Steel Total Flow800 mL/min. Flow A 584 mL/min. Flow B 216 mL/min. Temperature ° C. 25and 40

Batch 4.1

Ultrafiltration and 1× PBS exchange was performed on a On Labtop Systemthat has been fitted with 5×100K PES Slice cassettes. The system wascleaned in place with ethanol wash followed by water wash thenequilibrated with 1× PBS. Once equilibrated and with the vessel fullwith 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 300 RPM. After concentration of 1.5 L, the pumpwas increased to 400 RPM. The formulation was exchanged with 1× PBS. Thepermeate flow rate for the ultrafiltration step is shown in Table 43.

TABLE 43 Permeate flow rate Vessel Temperature ° C. 7 Pump SpeedPermeate flow rate (mL/min.) Initial 450   1 L 240 1.5 L 270   4 L 120  8 L 60

Batch 4.2:

Ultrafiltration and 1× PBS exchange was performed on a Labtop Systemthat has been fitted with 3×100K PES Slice cassettes. The system wascleaned in place with ethanol wash followed by water wash thenequilibrated with 1× PBS. Once equilibrated and with the vessel fullwith 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 300 RPM. The permeate flow rate data are shownin Table 44.

TABLE 44 Permeate flow rate Vessel Temperature 7° C. Pump Speed Permeateflow rate (mL/min.) Initial 700  1.5 L 450   3 L 330   4 L 200   5 L 190  6 L 130  7.5 L 100 8.25 L Final Concentration 90 PBS 1 L 105 PBS 3 L95 PBS 4 L 90

The final 400 mL was filtered (Sartopore 2 300 MN 5441307H5-OO) andparticle size and dispersity were measured. The data are shown in Tables45 and 46.

TABLE 45 Particle size, dispersity and lipid/siRNA ratio Z-AveLipid/siRNA Batch 4.1 (25° C.) (d · nm) PdI Ratio Initial 72.91 0.06511.4 Final UF 73.12 0.074 Permeate 77.3 0.063

TABLE 46 Particle size, dispersity and lipid/siRNA ratio Z-AveLipid/siRNA Batch 4.2 (40° C.) (d · nm) PdI Ratio Initial 79.45 0.03511.2 Final UF 76.62 0.062 10.7 Final UF Filtered 77.38 0.086 10.4

Example 18. Pump Speed and Permeate Flow Rate: 25% B and 31% B

On the system defined in Table 47, AD-1955 in 1 mmM Sodium Citrate wasmade at 1.044 mg/mL to target a Lipid/siRNA ratio of 10 at 25% B,representing the percent of the total flow rate for pump B, i.e., 25% B.For example, of the total flow of 800 mL/min, pump B is set to 25%giving 216 mL/min. B and 584 mL/min. A. Although the settings in thisexperiment using this particular tubing is 200/600 mL/min, the rates canbe used to tune the flow to achieve the desired Lipid/RNA ratio at theend.

The pumps were primed to waste. In this run, two formulations were made:(1) Batch 5.1 at 25% B (Theoretical lipid/RNA+10) and (2) Batch 5.2 at31% B (Theoretical lipid/RNA+14). For each, the formulation wascollected in a 10 L Bottle with 7 L 1× PBS (˜7.7 L total formulation).

TABLE 47 System Configuration A: Pump, Varian AD-1955 (1.25 L per batch)B: Pump, Varian AF-011 lipid mixture (0.46 L per batch) Inlet Tubing ID″Nat 0.062 Outlet Tubing ID″ Nat 0.08 TEE Stainless Steel Total Flow 800mL/min. Flow A 600 mL/min. (552 mL/min for 5.2) Flow B 200 mL/min. (248mL/min for 5.2) Temperature ° C. 25

Each formulation was filtered prior to UF with (Sartopore 2 300 MN5441307H5-OO) 0.45 μM+0.2 μm PES.

Batch 5.1

Ultrafiltration and 1× PBS exchange was performed on an On Labtop Systemthat has been fitted with 3×100K PES Slice cassettes. The system wascleaned in place with ethanol wash followed by water wash thenequilibrated with 1× PBS. Once equilibrated and with the vessel fullwith 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 450 RPM. The flow rate is shown in Table 48.

TABLE 48 Flow rate Vessel Temperature 7° C. Pump Speed 450 RPM Permeateflow rate (mL/min.) Initial 810 2.5 L 605   4 L 530   5 L 480   6 L 450  7 L 430   8 L Final Concentration 410 PBS 1 L 350 PBS 3.5 L 300 PBS 6L 280

The final 300 mL was filtered using the Sartopore filter (Sartopore 2300 MN 5441307H5-OO).

Batch 5.2

Ultrafiltration and 1× PBS exchange was performed on an On Labtop Systemthat has been fitted with 3×100K PES Slice cassettes. The system wascleaned in place with ethanol wash followed by water wash thenequilibrated with 1× PBS. Once equilibrated and with the vessel fullwith 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 450 RPM. The flow rate is shown in Table 49.

TABLE 49 Flow rate Vessel Temperature 7° C. Pump Speed 450 RPM Permeateflow rate (mL/min.) Initial 1000 2 L 650 4 L 500 6 L 430 8 L FinalConcentration 430 7 L 430 PBS initial 360 PBS 1 L 320 PBS 3 L 310 PBS 6L 220

The final 250 mL was filtered using the Sartopore filter (Sartopore 2300 MN 5441307H5-OO). The particles were measured and the lipid/RNAratios determined. The data are shown in Table 50.

TABLE 50 Particle size, dispersity and lipid/RNA ratio Z-Ave Lipid/siRNASample Name (d · nm) PdI Ratio Batch 5-1 Initial 9.94 Batch 5-1 UF 71.650.066 10.64 Batch 5-1 Final Filtered 71.67 0.031 10.60 Batch 5-2 Initial70.61 0.077 17.95 Batch 5-2 UF 75.51 0.062 14.22 Batch 5-2 FinalFiltered 72.69 0.082 14.95

Horiba analysis showed that some large particles in Batch 5.2 wereremoved by filtration.

Example 19. Salt Addition

In an effort to determine the effect of salt addition, NaCl was added toRNA solutions with mixing at 60° C. The system configuration was asdefined in Table 51.

Four batches were prepared: (1) Batch 6.1 with 10 mM NaCl, (2) Batch 6.2with 20 mM NaCl, (3) Batch 6.3 with 40 mM NaCl, and (4) Batch 6.4, noNaCl at 60° C. The particle size, dispersity and lipid/siRNA ratio weremeasured. The data are shown in Table 52.

TABLE 51 System configuration A: Pump, Varian AD-1955 B: Pump, VarianAF-011lipid mixture Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat0.08  TEE Stainless Steel Total Flow 800 mL/min. Flow A 600 mL/min. FlowB 200 mL/min. Temperature ° C. 25

TABLE 52 Effect of salt Z-Ave Lipid/siRNA Sample Name (d · nm) PdI Ratio6.1 initial 10 mM NaCl 76.57 0.079 6.4 6.2 initial 20 mM NaCl 68.960.083 11.1 6.3 initial 40 mM NaCl 70.73 0.081 11.2 6.4 initial no salt60° C. 77.89 0.075 11.1

Example 20. Pump Speed and Ultrafiltration Cassettes

On the system defined in Table 53, the pumps were primed to waste andthen the formulations were collected in 2×10 L Bottle with 8 L 1× PBS.The formulation was split into 5×5 L formulations for UF experiments.Four batches were investigated at various pump speeds and UF cassettes.The data are shown in Tables 54-57.

TABLE 53 System configuration A: Pump, Varian AD-1955 B: Pump, VarianAF-011lipid mixture Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat0.08  TEE Stainless Steel Total Flow 800 mL/min. Flow A 600 mL/min. FlowB 200 mL/min. Temperature ° C. 25

TABLE 54 Batch 7.1 Batch 7.1 Z-Ave 3 × 100K PES, 555 RPM (d · nm) PdI7.1 Mix 77.315 .047 7.1 UF 80.23 0.159 7.1 Fin Filter 80.5 0.109 7.1 FinFilter2x 76.33 0.075

TABLE 55 Batch 7.2 Batch 7.2 Z-Ave 3 × 300K PES, 555 RPM (d · nm) PdI7.2 Mix 77.315 0.047 7.2 UF 92.43 0.246 7.2 Fin Filter 92.5 0.192 7.2Fin Filter2x 85.32 0.188

TABLE 56 Batch 7.3 Batch 7.3 Z-Ave 3 × 100K PES, 450 RPM (d · nm) PdI7.3 Mix 77.315 0.047 7.3 UF 78.32 0.082 7.3 Fin Filter 80.41 0.035 7.3Fin Filter2x 79.38 0.061

TABLE 57 Batch 7.4 Batch 7.4 Z-Ave 3 × 100K PES, 550 RPM (d · nm) PdI7.4 Mix 77.315 0.047 7.4 UF 76.47 0.107 7.4 Fin Filter 82.39 0.057 7.4Fin Filter2x 79.79 0.086

Horiba analysis confirmed that the initial batch was good and UF at 550RPM created large particles. UF with the 300K cassettes was found toclog the cassettes producing particles of lipid/RNA ratios of between6-13.

Example 21. Ultrafiltration

On the system defined in Table 58, the pumps were primed to waste andthen the formulations were collected in 2×10 L Bottle with 8 L 1× PBS.

TABLE 58 System Configuration A: Pump, Varian AD-1955 B: Pump, VarianAF-011lipid mixture Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat0.08  TEE Stainless Steel Total Flow 800 mL/min. Flow A 600 mL/min. FlowB 200 mL/min. Temperature ° C. 25

Ultrafiltration and 1× PBS exchange was performed on a On Labtop Systemthat has been fitted with 3×100K PES Slice cassettes. The system wascleaned in place with ethanol wash followed by water wash thenequilibrated with 1× PBS. Once equilibrated and with the vessel fullwith 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 450 RPM. The solution was concentrated to 500mL, then exchanged with 5 L 1× PBS. Particles were measured 5 timeseach. The initial measurement and final average (n=5) are shown in Table59. From the data, it was clear that no change occurred in dispersityduring ultrafiltration.

TABLE 59 Averaged particle size Z-Ave Sample Name (d · nm) PdI Initialave. 69.018 0.0932 UF ave. 70.568 0.093

Example 22. Mixing Connector

On the system defined in Table 60, the pumps were primed to waste andthen the formulations were collected in 50 mL Falcon Tubes with 25 mL 1×PBS.

TABLE 60 System configuration A: Pump, Varian AD-1955 B: Pump, VarianAF-011lipid mixture Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat0.08  TEE Stainless Steel T and Plastic Y Total Flow 800 mL/min. Flow A600 mL/min. Flow B 200 mL/min. Temperature ° C. 25

Four small experiments were performed collecting 25 mL of formulationinto a 50 mL Falcon tube with 25 mL 1× PBS. The data are shown in Table61. From the data, it can be determined that particle size anddispersity varies based on the type of mixing connector, at least in thesmall sample sizes.

TABLE 61 Connector geometry Z-Ave Sample Name (d · nm) PdI Batch 11.1,Normal T Average 71.53 0.075 Batch 11.2, Cross T Average 86.83 0.089Batch 11.3, Symmetric Y Average 69.92 0.050 Batch 11.4, Asymmetric YAverage 80.92 0.102

Example 23. Mixing Connector: Study 2

On the system defined in Table 62, the pumps were primed to waste andthen the formulations (1.25 L siRNA and 0.41 L lipids) were collected in10 L Bottle with 8 L 1× PBS.

TABLE 62 System configuration A: Pump, Varian AD-1955 B: Pump, VarianAF-011 lipid mixture Inlet Tubing ID″ Nat 0.062 Outlet Tubing ID″ Nat0.08  TEE Stainless Steel T and Plastic Y Total Flow 800 mL/min. Flow A600 mL/min. Flow B 200 mL/min. Temperature ° C. 25

Two batches were prepared; 12.1 using the symmetrical “Y” for mixing and12.2 using the stainless steel “T” for mixing. Each batch (1.25 LAD-1955 solution) was collected in a 10 L bottle prepped with 8 L 1×PBS. Cloudiness was noticed in the permeate with Batch 12.1 and theprocess was stopped for this batch.

Both batches were filtered using a Sartopore 0.45 μm to 0.2 μm in-lingfilter. Ultrafiltration and 1× PBS exchange was performed on an OnLabtop System that has been fitted with 3×100K PES Slice cassettes. Thesystem was cleaned in place with ethanol wash followed by water washthen equilibrated with 1× PBS. Once equilibrated and with the vesselfull with 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 450 RPM.

When all formulation was in the vessel, immediately began diafiltrationwith 10 L bottle prepped with 8 L 1× PBS by moving the feed tube to abottle with 10 L 1× PBS. After diafiltration, reduced the pump speed to300 RPM and concentrate to approximately 500 mL. The concentratedproduct was collected. Particles were measured for size and dispersity.

Cloudiness was noticed in the permeate with Batch 12.1 and the processwas stopped for this batch.

TABLE 63 Particle size and dispersity: connector study 2 Z-Ave SampleName (d · nm) PdI 12.1 UF Ave. 65.13 0.107 12.1 mix Ave. 72.60 0.06112.1 UF Filter Ave. 64.48 0.102 12.2 mix Ave. 72.50 0.072 12.2 UF1Average 70.42 0.064 12.2 UF2 Average 69.87 0.081

Example 24. Mixing Connector: Study 3

On the system defined in Table 64, the pumps were primed to waste andthen the formulations were collected in 10 L Bottle with 8 L 1× PBS.

TABLE 64 System configuration Batch 13 Batch 14 Mix 1.5 L siRNA Mix 0.8L siRNA Item and 0.5 L lipids and 0.26 L lipids. A: Pump, Varian AD-1955AD-1955 B: Pump, Varian AF-011 lipid AF-011 lipid mixture mixture InletTubing ID″ Nat 0.062 Nat 0.062 Outlet Tubing ID″ Nat 0.08  Nat 0.08  TEEStainless Steel T Plastic Y Total Flow 800 mL/min. 800 mL/min. Flow A600 mL/min. 600 mL/min. Flow B 200 mL/min. 200 mL/min. Temperature ° C.25 25

Both batches were filtered using a Sartopore 0.45 μm to 0.2 μm in-lingfilter. Ultrafiltration and 1× PBS exchange was performed on an OnLabtop System that has been fitted with 3×100K PES Slice cassettes. Thesystem was cleaned in place with ethanol wash followed by water washthen equilibrated with 1× PBS. Once equilibrated and with the vesselfull with 1× PBS, the formulation was added by vacuum and concentratedstarted at pump speed of 450 RPM. When all formulation was in thevessel, immediately began diafiltration with 10 L 1× PBS by moving thefeed tube to a bottle with 10 L 1× PBS. After diafiltration, reduced thepump speed to 300 RPM and concentrate to approximately 500 mL. Theconcentrated product was collected. Particles were measured for size anddispersity.

TABLE 65 Particle size and dispersity; connector study 3 Z-Ave SampleName (d · nm) PdI 13 T MIX Ave. 79.16 0.063 13 T UF Ave. 73.61 0.086 14Y MIX Ave. 70.32 0.084 14 Y UF Ave. 69.93 0.086

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1. A method of preparing a formulation comprising lipid nanoparticlescomprising an RNAi agent payload comprising; (a) mixing a first solutionwith a second solution in a mixing connector, wherein (i) said firstsolution comprises an ethanolic solution comprising one or more lipidsand having a total lipid concentration of approximately 30 mg/mL, and(ii) said second solution comprises citrate buffered aqueous solutioncomprising one or more RNAi agents and having an RNAi agentconcentration of approximately 1 mg/mL and a pH of between 3 and 6, and(b) diluting the mixture produced in (a) in a vessel containing a buffersolution thereby producing a formulation comprising lipid nanoparticlescomprising an RNAi agent payload; wherein the linear flow rate of saidfirst solution into the mixing connector is approximately 606,267 cm/hand the linear flow rate of said second solution into the mixingconnector is approximately 1,818,801 cm/h and the volume ratio of saidfirst solution to said second solution is approximately 1:3.
 2. Themethod of claim 1 wherein the buffer solution of (b) is phosphatebuffered saline (PBS).
 3. The method of claim 2, wherein the vesselcontains sufficient PBS to dilute the mixture resulting from step (a) bya factor of between 4 and 10 fold.
 4. The method of claim 3, wherein thefactor is 5 fold and the final concentration of ethanol in theformulation is equal to or less than 5%.
 5. The method of claim 1,further comprising; (c) ultrafiltration of said formulation comprising;(i) concentration of said formulation such that the lipid nanoparticleconcentration is increased by a factor of between 1 and 10 fold, (ii)diafiltration of the concentrated formulation of (i) using at least 10volume exchanges with buffer solution, wherein the ethanol concentrationis reduced to less than 1%, and (iii) concentration of the filteredformulation of (ii) to produce an RNAi agent concentration of between2.5 and 3 mg/mL.
 6. The method of claim 5, further comprising; (iv)adjusting the RNAi agent concentration of the formulation of (iii) to aconcentration of 2 mg/mL by the addition of PBS. 7.-9. (canceled) 10.The method of claim 1, wherein the total lipid to RNAi agent w/w ratiois between 10:1 and 14:1 based on the total lipid concentration of saidfirst solution and the RNAi agent concentration of said second solutionas determined prior to mixing using HPLC.
 11. A system for themanufacture of a formulation comprising lipid nanoparticles comprisingan RNAi agent payload comprising; (a) a first reservoir providing afirst solution, wherein said first solution is an ethanolic solutioncomprising one or more lipids, (b) a second reservoir providing a secondsolution, wherein said second solution is a buffered aqueous solutioncomprising one or more RNAi agents, (c) a first pump, operably connectedto said first reservoir and configured to regulate the flow of saidfirst solution at a linear flow rate of between 303,133 and 909,400cm/h, (d) a second pump, operably connected to said second reservoir andconfigured to regulate the flow of said second solution at a linear flowrate of between 1,515,667 and 2,121,934 cm/h, (e) a mixing connectorcomprising at least a first inlet, a second inlet and an outlet, whereinsaid first inlet receives flow from said first pump and said secondinlet receives flow from said second pump, (f) at least one heatexchanger operably connecting each of said first and said second pumpsto said inlets of the mixing connector, respectively, and (g) a vesselfor receiving effluent from the outlet of said mixing connector.
 12. Thesystem of claim 11, further comprising an ultrafiltration systemconfigured to receive effluent from said vessel, said effluentcomprising the lipid nanoparticle formulation.
 13. The system of claim12, wherein the ultrafiltration system comprises a rotary lobe pump or adiaphragm pump which allows for lipid nanoparticle formulation retentatecirculation and permeate transport across a membrane filter. 14.(canceled)
 15. The system of claim 13, wherein the filter is apolyethersulphone membrane.
 16. The system of claim 13, wherein thetransmembrane pressure across the membrane filter is between 5 and 15psi.
 17. The system of claim 13, wherein the permeate flow rate isbetween 50 and 400, 60 and 300 or 100 and 200 liter/m²/h.
 18. The systemof claim 11, further comprising; (g) at least one filter or filtrationdevice operably engaged in front of each of said first and said secondreservoirs.
 19. (canceled)
 20. (canceled)
 21. The system of claim 11,wherein the planar angle between said first inlet and said second inletis between 5 and 180 degrees.
 22. The system of claim 21, wherein theplanar angle between said first and said second inlet is 120 degrees.23. The system of claim 11, wherein the mixing connector comprises 3, 4,5, 6, 7 or 8 inlets, each configured to receive flow from either saidfirst pump or said second pump.
 24. The system of claim 23, wherein theinlets of the mixing connector are positioned equidistant from oneanother about a central axis.
 25. (canceled)
 26. (canceled)
 27. A lipidnanoparticle formulation comprising an RNAi agent payload prepared bythe process of claim
 1. 28. The method of claim 1, wherein the RNAiagent is selected from the group consisting of siRNA, dsRNA, miRNA, andnucleotide sequences encoding the same.